Human T-cell leukemia viruses type 1 (HTLV-1) is notorious for being difficult to study in cell culture, which has prohibited a rigorous analysis of how these viruses replicate in cells, including the steps involved in retrovirus assembly. The details for how retrovirus particle assembly occurs are poorly understood even for other more tractable retroviral systems. This proposal seeks funding to investigate two questions related to Gag trafficking in living cells using an exciting new biophysical approach. We will apply the novel single-molecule technology of fluorescence fluctuation spectroscopy (FFS) in living cells to investigate 1) HTLV-1 Gag oligomerization of wt Gag and mutants that have defects in binding proteins involved in virus budding as well as Gag mutants that have RNA packaging defects, and 2) Gag mobility and binding affinity to membrane. The results from these proposed studies should provide further insight into fundamental aspects of HTLV-1 and retrovirus particle assembly. PUBLIC HEALTH RELEVANCE: Fundamental studies of HTLV-1 assembly will lead to detailed information about these processes that will be useful for a better understanding of how these viruses replicate in cells.